REVIEW
Mycetoma laboratory diagnosis: Review
OPEN ACCESS
Citation: Ahmed AA, van de Sande W, Fahal
AH (2017) Mycetoma laboratory diagnosis:
Review article. PLoS Negl Trop Dis 11(8):
e0005638.
https://doi.org/10.1371/journal.pntd.0005638
Editor: Archie C. A. Clements, University
of Queensland, AUSTRALIA
Published: August 24, 2017
Copyright: © 2017 Ahmed et al. This is an
open access article distributed under the
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distribution, and reproduction in any medium,
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Funding: The authors received no specific
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Competing interests: The authors have declared
that no competing interests exist.
PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005638 August 24,
2017
article
Amel Altayeb Ahmed1, Wendy van de Sande2, Ahmed Hassan Fahal1*
1 The Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan, 2 Department of Medical
Microbiology & Infectious Diseases, Erasmus MC, University of Rotterdam, Rotterdam, the Netherlands
* ahfahal@hotmail.com, ahfahal@uofk.edu
Abstract
Mycetoma is a unique neglected tropical disease caused by a substantial number of
micro- organisms of fungal or bacterial origins. Identification of the causative organism
and the dis- ease extension are the first steps in the management of the affected
patients and predicting disease treatment outcome and prognosis.
Different laboratory-based diagnostic tools and techniques were developed over the
years to determine and identify the causative agents. These include direct microscopy
and cytological, histopathological, and immunohistochemical techniques in addition to the
classi- cal grain culture. More recently, various molecular-based techniques have joined
the myce- toma diagnostic armamentarium. The available mycetoma diagnostic
techniques are of various specificity and sensitivity rates. Most are invasive, time
consuming, and operator dependent, and a combination of them is required to reach a
diagnosis. In addition, they need a well-equipped laboratory and are therefore not field
friendly.
This review aims to provide an update on the laboratory investigations used in the
diag- nosis of mycetoma. It further aims to assist practising health professionals dealing
with mycetoma by outlining the guidelines developed by the Mycetoma Research
Centre, Uni- versity of Khartoum, WHO collaborating centre on mycetoma following a
cumulative experi- ence of managing more than 7,700 mycetoma patients.
Introduction
Mycetoma is a devastating chronic subcutaneous granulomatous inflammatory disease caused
by several true fungi and bacteria, and hence it is classified as eumycetoma and actinomyce-
toma, respectively [1,2]. The disease is characterised by numerous deformations and disabili-
ties, high morbidity, and in its late stage it is potentially fatal. Mycetoma is endemic in the so-
called “mycetoma belt” that includes various countries across the world, but it is reported
extensively from Sudan, Mexico, and India [3,4].
The triad of a painless subcutaneous mass, multiple sinuses, and discharge that contains
grains of different colours, sizes, and consistency is characteristic of mycetoma [2]. Late pre-
sentation of the majority of patients is the norm, and the explanation is multifactorial, with
reasons including the painless nature of the disease, patients’ low socioeconomic status and
health education, and scarcity of health facilities in endemic regions (Fig 1) [3,5,6,7].
1 / 17
 Fig 1. Photography showing the mycetoma triad of mass, multiple discharging sinuses, and black grains.
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Mycetoma is classified as eumycetoma and actinomycetoma, and they are caused by

a considerable number of microorganisms of both fungal and bacterial origin,
respectively (Table 1).

Table 1. The common different mycetoma causative organisms.

Grain color Causative organisms Diagnosis
Eumycetoma
Black grains Madurella spp. Histopathological examination
Leptosphaeria spp. PCR
Curvularia spp.
Exophiala spp.
Phaeoacremonium spp.
Phialophora verrucosa
Pyrenochaeta mackinnonii
P. romeroi
Pale, white, yellow grains Pseudallescheria boydii (Scedosporium apiospermum)
Acremonium spp.
Aspergillus spp.
Actinomycetoma
Pale, white, yellow grains Actinomadura madurae, Nocardia spp. Histopathological examination
Yellow to brown grains Streptomyces spp. PCR
Red to pink grains A. pelletierii
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 Hence, proper treatment of mycetoma requires adequate and accurate diagnosis of the
causative organisms. Currently, a series of investigations are available to establish the
diagnosis of mycetoma [8]. Most of these investigations are invasive, time consuming, and
require good personal experience. In most instances, a combination of these investigations is
required in a well-equipped laboratory to reach a diagnosis [8]. The aim of this article is to
discuss the pros and cons of the available laboratory-based diagnostic investigations in order
to assist treating clinicians with requesting appropriate investigations.

Mycetoma grains
Grains are crucial to establish the diagnosis of the causative organism. Although the grains’
morphological characteristics may provide a rapid provisional identification of the aetiological
agent, in some instances they may be deceiving [1,4,5,8]. Grains have different morphological
features; their size varies from microscopic to 1–2 mm in diameter. Madurella mycetomatis
and A. madurae have large grains, whereas Nocardia brasiliensis, N. cavae, and N. asteroids
grains are small in size [8–11]. Their colour is variable, ranging from black, yellow, white, or
red to pale. Most of eumycetoma causative organisms produce black or pale grains and rarely
yellow, and actinomycetoma commonly is caused by organisms that produce yellow, white, or
red grains [10]. The consistency of most grains is soft, but Streptomyces somaliensis and M.
mycetomatis are quite hard (Fig 2) [1].
Grains are commonly obtained by deep surgical biopsies under aseptic conditions to
avoid contamination. Grains obtained from open sinuses are commonly not viable and
often con- taminated [10].

Fig 2. Photography of surgical biopsy showing a well-encapsulated eumycetoma lesion with numerous black
grains.
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 The surgical biopsies must be handled immediately by the laboratory technologist in the
surgical theatre. The biopsy should be divided into 2 parts; 1 part for the grains culture and the
other for histopathological examinations. The former is placed in normal saline, while the lat-
ter is placed in 10% formal saline.

Grains direct microscopy

Direct microscopic examination of grains obtained from the sinuses’ serosanguineous dis-
charge is the fastest means of making a presumptive diagnosis of the mycetoma causative
organisms, but it lacks accuracy. The grains can be directly examined under light microscope
using 10% potassium hydroxide (KOH), which digests the mucus and keratin, thus providing
a clear background [9,10, 11,12]. Direct grain microscopy can rule out actinomycotic
causative agents. However, it cannot discriminate between certain organisms such as M.
mycetomatis and Trematosphaeria griesia. Therefore, this procedure is not specific enough
to make a defini- tive diagnosis of mycetoma and should be supplemented by additional
characteristic features identification. In addition to the 10% KOH, Parker ink can be used to
examine the serosangui- neous discharge containing grains microscopically.
The crushed grains are placed on a glass slide and covered with a coverslip. Two or 3
drops of the stain are applied to the slide edge and allowed to infiltrate under the slip. The
prepara- tion is then examined under light microscope for the presence of the hyphae and
spores. They usually take up a dark blue colour in a light blue cellular background.
Actinomycetes under the microscope usually show branching filaments, abundant aerial
mycelium, and long chains of spores (Fig 3).
Fabric fibres can cause diagnostic confusion, as they can also take up the stain, but
they are often out of the tissue plane, larger than the hyphae, irregular in diameter, and
often have an irregular spiral configuration [9,10].

Fig 3. KOH wet mount direct microscopic examination of M. mycetomatis grains showing its hyphal
structure.
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 Several histochemical staining techniques are used for rapid identification of mycetoma
causative agents from culture. Gram staining and the Ziehl–Neelsen (ZN) staining
technique are the commonest in use. Actinomycetoma causative organisms are gram-
positive, while eumycetoma causative organisms are gram-negative [9,10,12]. The
actinomycetes consist of fine, branching filaments, about 1 micron thick, whereas the
eumycetes grains are composed of septate hyphae 4–5 microns thick. The ZN staining
technique is superior in discriminating between actinomycotic agents; Nocardia spp. are ZN
positive, whereas A. madurae and S. somaliensis are ZN negative [9, 10,12].

Grains culture
A sizeable number of grains are needed to culture the causative agents of mycetoma. They
must be soaked and stored in saline for culture, washed several times with normal saline, and
inoculated onto suitable culture media in sterilised conditions using either a safety cabinet or a
flame-sterilised area. Modified Sabouraud agar supplemented with 0.5% yeast extract, blood
agar, brain–heart infusion agar, and Lo¨wenstein agar are the commonly recommended
media.
Antibiotic free culture media is required for the isolation of actinomycetes, while
the eumycetes culture must contain antibiotics. The commonly used antibiotics are
penicillin G (20 U/ml), gentamicin sulphate (400 μg/ml), streptomycin (40 μg/ml), or
chloramphenicol (50 μg/ml).
The mycetoma causative organisms can be identified by their textural description and
mor- phological and biological activities in pure culture. The biological activity may
include acid fastness, optimal temperature, proteolytic activity, utilisation of sugars, and
nitrogenous com- pounds [12].
Nocardia typically produce substrate and aerial hyphae that look like rods and coccoids.
Streptomyces form a yellowish substrate mycelium and lack aerial hyphae, while
identification of Madurella is mainly based on the morphology of the fruiting bodies and
morphology of the colonies (Fig 4) [9,10,12,13].
The phenotypic characteristics of the causative organisms are important for their identifica-
tion. These include the production of β-glucuronidase; degradation of adenine, casein, and
hypoxanthine; growth on adonitol; aesculin hydrolysis; glycerol; glycogen; D-raffinose; L-
rhamnose; D-turanose; D-xylose; and L-aspartic acid. The latter is a sole carbon source and

Fig 4. Photograph showing M. mycetomatis growth in Sabouraud agar media.

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 plays a role in the identification of pathogenic Streptomyces spp. A. madurae was found to be
positive for α-glucosidase and negative for N-acetyl-β-glucosaminidase [9,10,12].
In view of the limited information available on phenotypic properties and assimilation pat-
terns for the identification of eumycetoma agents, a new system, the API 20C AUX kit, was
recently introduced and was able to identify M. fahalii, M. pseudomycetomatis, and M. tropi-
cana [14].
Differentiation of the various dematiaceous fungi on the basis of morphology is sometimes
difficult and time consuming, and culture usually takes about 3 weeks to give an accurate
result. However, the culture technique is time consuming, and accidental contamination may
give a false positive result. It also requires experience to identify the causative organisms
[3,4,12,15].

Mycetoma cytological identification

Fine-needle aspiration cytology (FNAC) with cell blocks and imprint cytology techniques for
mycetoma were described [16,17]. Fine-needle aspirations under aseptic conditions is required
to identify the causative agent of mycetoma and the tissue reaction against it. In this technique,
a needle attached to a syringe is inserted into the suspected mycetoma lesion and aspirated
under negative pressure. It should be performed in at least 3 different directions (Fig 5). After
fixation, the cell blocks are processed and stained as is done with the paraffin sections. The
smears or the sections can be examined microscopically.
Mycetoma has characteristically distinct cytological features, characterised by the presence
of suppurative granulomas surrounding the characteristic grains of the causative organism.
The granuloma consists of neutrophilic infiltrate in close contact with and infiltrating the

Fig 5. Photograph showing the fine-needle aspiration cytology collection technique.

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grains. It is surrounded by palisading histiocytes beyond which there is a mixed inflammatory
infiltrate comprising lymphocytes, plasma cells, eosinophils, macrophages, and fibrosis.
Multi- nucleated giant cells are frequently encountered in the granuloma.
In smears stained with haematoxylin and eosin (H&E), the M. mycetomatis grains
appeared rounded or oval in shape and black with a green tinge of colour or occasionally
brownish. Two types of M. mycetomatis grains can be identified in cytological smears: the
solid granular type, which is the commonest, and the vesicular type. The septate hyphae are
not identified in the first type because they are embedded in a hard, brown cement matrix.
The vesicular type con- sists of swollen fungal cells and are seen as vesicles (Fig 6).
Actinomycetes grains are homogeneously eosinophilic in H&E. In Geimsa-stained smears,
the grain appears homogeneously blue in the centre, while in the periphery it consists of fine
granules and radiating pink filaments. The A. pelletierii grain is more eosinophilic in H&E as
compared to S. somaliensis, and it is semilunar in shape, as seen in histology.
The cytological smears can differentiate mycetoma from other subcutaneous lesions and
can identify the mycetoma causative agents. The technique is simple, quick, economical, and
can be used for sample collection in epidemiological surveys and culture. The presence of
grains in the cytological smear is mandatory to reach a diagnosis. The technique is noted to be
painful in some patients and may induce infection and cellulitis in the area [16,17].
EL Hag and colleagues in 1995 studied a group of 14 patients with different types of
myce- toma lesions using the FNAC technique. The findings from the cytological smears
were com- parable to those observed in histological sections. They concluded that this
technique is useful for the routine diagnosis of mycetoma in epidemiologic surveys and for
material collection [16]. Yousif and colleagues in 2009 used FNAC and the cell block
technique in the diagnosis of 230 patients with different types of mycetoma, and they reported
sensitivity rates of 87.5% and 85.7% for eumycetoma and actinomycetoma identification,
respectively [17].
In summary, simple and inexpensive cytological techniques such as FNAC and imprint
smears that employ routine H&E, May–Gru¨nwald–Giemsa, Papanicolaou, and periodic
acid– Schiff (PAS) stains on cytological specimen usually lead to rapid diagnosis of
mycetoma, par- ticularly in remote, endemic regions.

Fig 6. Photomicrograph showing M. mycetomatis and inflammatory infiltrate in a cytological smear.

Haematoxylin and eosin x 40.
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Histopathological and histochemical techniques
Surgical biopsies are commonly obtained by wide local excision or deep incisional biopsy,
and currently a Tru-Cut needle biopsy is in use. Surgical biopsies taken under local
anaesthesia are painful and usually yield inadequate specimens and have to be avoided [6].
The initial step is the fixation of biopsy samples in suitable fixatives such as 10%
formal saline. The tissue processing then follows several steps: dehydration by different
concentra- tions of ethanol, clearing by Xylene, impregnation and embedding by using
paraffin wax (a 2- to 5-micron-thick section obtained by microtome), and finally staining
with different staining techniques [12,18,19].
H&E is the stain for primary identification of the causative agent and the tissue reaction.
Special stains usually follow for accurate identification of certain organisms and the cell com-
ponents such as proteins, lipids, carbohydrates, and minerals that can be associated with the
disease.
The special stains commonly in use are Grocott’s hexamine silver, PAS, Masson–Fontana
stain, Perl’s Prussian blue, von Kossa’s stain, formalin-induced fluorescence, and Schmorl’s
stain. Modified bleaching technique is also in use [12,18,19].
Actinomycete grains can be identified by Gram and ZN stains [12,18,19]. H&E, PAS, Gro-
cott’s methenamine silver, and Gridley are the most useful stains for detecting hyphae and
chlamydospores in eumycetes grains [12, 18,19].
Under microscopy, the fungal structures are broad, septate, and branching hyphae with
large swollen cells at the edge. The hyphae may be hyaline or pigmented. Grain cement may
or may not be seen, and if present, it may be compact or loose. These characteristics are
useful for identification but may not be used for definitive diagnosis [18,19].
The M. mycetomatis grains are large, ranging from 0.5 to 3 mm; appear rounded, oval, or
trilobed; and consist of intertwining hyphae embedded in interstitial brownish cement. The
cement contains melanin, heavy metals, proteins, and lipids [18,19]. The grain can consist of
filamentous or vesicular types. Brown septate and branched hyphae that may be slightly more
swollen in the periphery are indicative of a filamentous grain, while unusually large cells that
look like vesicles predominate in the vesicular type [18,19,20].
The host tissue reaction against the mycetoma causative organisms is distinctive, [21,22].
There are 3 types of tissue reactions.

Type I reaction
In this reaction, the grains are surrounded by a layer of polymorphonuclear leukocytes,
with neutrophils closely attached to the surface of the grain or invading the substance of
the grain. This is surrounded by a layer of cells consisting of plasma cells, macrophages,
lymphocytes, and a few neutrophils in a granulation tissue. Layers of fibrin usually
surround the venules and the capillaries, and the outermost layer of the lesion contains
fibrous tissue (Fig 7).

Type II reaction
In this reaction, the macrophages and multinucleated giant cells replace most of the
neutro- phils. Fragments of the destroyed grains are commonly seen within the
multinucleated giant cells (Fig 8).

Type III reaction

This reaction is commonly characterised by well-organized epithelioid granulomas containing
Langerhans giant cells, and usually no grains are seen (Fig 9).
Fig 7. Photomicrograph showing a M. mycetomatis grain with a type I tissue reaction. Haematoxylin
and eosin x 10.
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The technique is remarkable, as it does not require aseptic technique or need an

inflexible time frame. However, accurate identification of certain organisms proves to
be difficult, it lacks the culture precision, and it takes several days to deliver the results.
A deep biopsy that contains grains is always needed to establish a diagnosis (Fig 10).

Serodiagnosis
Over the years, different serological tests and assays were used for the diagnosis of
mycetoma. These included immunoblots, indirect haemagglutination assays (IHAs),
immunodiffusion (ID), counterimmunoelectrophoresis (CIE), and ELISA (Fig 11) [8,23–
27].

Fig 8. Photomicrograph showing M. mycetomatis and a type II tissue reaction. Haematoxylin and
eosin x 10.
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Fig 9. Photomicrograph showing a type III tissue reaction. Haematoxylin and eosin x 10.
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Salinas-Carmona and his associates reported on the use of ELISA for the serological diagno-
sis of N. brasiliensis, the most common agent causing actinomycetoma in Mexico [23]. This
study revealed a higher incidence of antibodies in patients with active disease without cross-
reactions with Mycobacterium leprae and M. tuberculosis. This has been useful in cases in
which identification of the aetiological agent in culture was not possible.
For eumycetoma agents, serological assays have been developed only for M.
mycetomatis and P. boydii. For P. boydii, both an ID assay with crude antigens and an IHA
were developed. The ID assay and CIE test for M. mycetomatis were most widely applied
using crude cytoplas- matic antigens [24,25]. CIE was superior to ID since lower antibody
titres were found. Unfortu- nately, since crude antigen preparations were used, cross-
reactivity occurred and reproducibility was low [24,25]. The only serological assays
performed with pure antigens of M. mycetomatis were an ELISA based on the recombinant-
produced translationally controlled tumour protein

Fig 10. Photomicrograph showing M. mycetomatis hyphae and cement substances that are
positive for calcium stained with von Kossa stain.
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Fig 11. Photograph showing a counterimmunoelectrophoresis test with positive bands.
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(TCTP) and the Luminex assays based on TCTP, fructose-bisphosphate aldolase (FBA), and pyru-
vate kinase (PK) [27,28]. Although patients had higher levels of antibodies, the same levels were
also detected in healthy controls, making the techniques unsuitable as diagnostic tools [27,28].
ELbadawi and associates in 2016 reported on the 75% sensitivity and 95% specificity of
immu- noblotting of M. mycetomatis cytoplasmic antigen from molecularly identified cultures
[29].
It is clear that these serodiagnostic tests have many limitations, which include the tedious
and lengthy preparation of antigens, the fact that the antigens are crude and not standardised,
and the cross-reactivity between different mycetoma causative organisms.

Molecular-based identification methods

Chemical methods, while effective in distinguishing between genera of actinomycetes, are
laborious and time consuming. They are complemented and replaced by systematic molecular
procedures, notably the 16S rRNA gene sequencing studies [19,30,31]. Other molecular meth-
ods championed for this purpose include PCR coupled with restriction endonuclease analyses
of PCR products, PCR-based randomly amplified polymorphic DNA fingerprinting, and
Curie-point pyrolysis mass spectrometry [32–36]. Such studies allow more accurate classifica-
tion of strains previously misclassified.
For eumycetoma, various molecular techniques have been used to identify the causative
agents, and all are based on the identification of the internal transcribed spacer (ITS). In order
to identify all fungal mycetoma causative agents, the ITS regions are usually amplified with pan-
fungal primers and sequenced [34–39]. Identification is based on comparing the resulting
sequence with sequences already present in GenBank. Using this approach, several studies
reported that a number of causal agents for eumycetoma are underspeciated, as exemplified by
the identification of 3 new Madurella species (M. fahalii, M. tropicana, and M.
pseudomycetoma- tis) as well as Pleurostomophoraochracea , a eumycetoma that produces yellow
grains [40,41].
For M. mycetomatis, Ahmed and colleagues developed a species-specific PCR (Fig 12)
[34]. This PCR–restriction fragment length polymorphism (RFLP) analysis showed strict
homo- geneity between M. mycetomatis isolates, [34] and it can be used to identify the
causative agent
not only from clinical material but also from environmental samples [42].
Molecular typing of the causative agents can also be performed. For M. mycetomatis,
vari- ous methods have been used, including restriction endonuclease analyses (REA),
random amplified polymorphic DNA (RAPD), and amplification fragment length
polymorphism (AFLP) [34–39]. Although results with RAPD are variable, REA and AFLP
were able to differ- entiate M. mycetomatis isolates from different countries or even
within a country. Certain AFLP types were associated with the origin of the strain or the
size of the lesion [34–39].
The genetic variability identification of mycetoma causative organisms is a more
stable approach than using methods based on phenotypic criteria. The molecular
techniques based
Fig 12. Showing Lane 1 contain a 100 bp DNA ladder. Lanes 2 to 4 show the PCR products for three
samples which were negative for Madurella mycetomatis. Lane 5 to 8 shows the PCR products for four
samples which were positive for Madurella mycetomatis. Lane 10 was a positive control; lane 11 was a
negative control.
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on genetic variation are analyses of electrophoretic karyotype differences and RFLPs using gel
electrophoresis or DNA–DNA hybridization [34–38].
Prior to DNA extraction, the fungi are usually subcultured on Sabouraud agar and
incu- bated at 37˚C for 3 weeks. The mycelia are scraped from the culture medium and
homogenised with sterile pestles and mortar. The homogenised mycelia must then be
snap frozen in liquid nitrogen, thawed and refrozen twice, and rehomogenised in 2 ml of
lysis buffer containing 4 M guanidiniumisothiocyanate, 0.1 M Tris-HCl (pH 6.4), 0.2 M
EDTA, and 0.1% Triton X-
100. Then DNA should be purified by Celite affinity chromatography [12].
The molecular identification of the causal agents of eumycetoma is mainly based on the
ITS that is located between the 18S and 28S genes. The causative agents can be identified to
the spe- cies level by molecular techniques [34–38].
Using a phylogenetic approach, de Hoog and colleagues studied the natural habitat of
Madurella species. Four species of Madurella were included in a large data set of species of
Chaetomidium, Thielavia, Chaetomium, and Papulaspora using sequences of the universal
fun- gal barcode gene rDNA ITS and the partial LSU gene sequence. They demonstrated that
Madurella species are nested within the Chaetomiaceae family [43].
Abdulla and associates in 2003 collected 38 different M. mycetomatis isolates and
analysed the samples by PCR using 20 different primer species. Their result showed a
complete lack of DNA fingerprint variation among the various isolates, and they
concluded that there is little genetic variation among clinically relevant M.
mycetomatis strains from Sudan [42].
In 2005, van de Sande described the genotyping of M. mycetomatis by selective AFLP
and subtype correlation with the geographical origin and lesion size to discriminate
between the strains [39].
Rolling circle amplification (RCA) uses species-specific padlock probes and isothermal
DNA amplification, has high specificity and simplicity, and is of low cost. Ahmed and col-
leagues in 2013 used this technique for the identification of Falciformispora senegalensis,
Falciformispora tompkinsii, M. fahalii, M. mycetomatis, M. pseudomycetomatis, M. tropicana,
Medicopsis romeroi, and T. grisea in a sample of 62 isolates, and the technique produced
100% specificity with no cross-reactivity or false results [37].
 Application of isothermal amplification techniques for identification of M. mycetomatis
was used, and the method was found to be reliable and easy to operate. It therefore has the
poten- tial to be implemented in areas where mycetoma is endemic. The techniques may be
expanded to detect fungal DNA from environmental samples [38].
In general, molecular-based techniques for mycetoma causative organisms are attractive, as
they can identify the organism to the species level and thus guide the optimum and appropriate
treatment. However, most are expensive, are not field friendly, and are not available in
endemic areas [34–39].
Genome sequencing is a new insight into the diagnosis of mycetoma, providing data about
the biology and the pathogenicity of the fungi by comparing the genome to other fungi.
M. mycetomatis mm55 was isolated in 1999 at the Mycetoma Research Centre, Khartoum,
Sudan, from an extensive foot mycetoma in a 22-year-old male patient. The strain was isolated
by direct culture of the black grains obtained by a deep biopsy and identified by morphology
after PCR-RFLP and sequencing of the ITS region. Strain mm55 was sequenced and identified
by Smit and colleagues in 2016 [44].
Lucio Vera-Cabrera and associates in 2014 reported on the draft genome sequence of a
member of the Thermomonosporaceae family, A. madurae LIID-AJ290, isolated
from a human case of mycetoma. The assembly contains 10,308,866 bps [45].

Conclusion
In conclusion, accurate identification of the mycetoma causative agent is a prerequisite for the
treatment of the disease. Different diagnostic tools are in use to ensure accurate and precise
diagnosis of mycetoma. Each method or technique has its advantages and disadvantages, and
their specificity and sensitivity are variable. Many factors influence the use of a technique,
among which are the type of the lesion, availability of well-equipped laboratory and expert

Fig 13. Flow chart for the diagnosis of mycetoma.

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staff, and technique cost. In almost all cases, a combination of techniques and methods is
required to reach a correct diagnosis of mycetoma. This is a call for an urgent need for simple,
accurate, reliable, cost-effective, and field-friendly diagnostic techniques.
In general, the current recommended protocol in endemic areas with meagre resources is
to start with FNAC; if the FNAC results are negative, then perform a deep surgical biopsy to
obtain grains for culture and conduct a histopathological examination. When available, appro-
priate molecular techniques molecular identification can be requested (Fig 13).

Key learning points

• An ideal diagnostic tool for mycetoma is lacking.
• A combination of techniques is always required to reach a diagnosis.
• The direct microscopy technique is rapid but lacks specificity.
• Cytological examination of a cytological smear is a rapid and simple tool.
• The histopathological technique can give accurate results provided that the
grains are available in the tissue section.
• Grain culture in sterile conditions and in expert hands has high yield.
• Molecular techniques in well-equipped centres provide an accurate
identification of the causative agents of mycetoma.
• There is an urgent need for simple, accurate, reliable, cost-effective and field-
friendly diagnostic tests.

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